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Background Safety, environmental and economic setbacks are driving industries to find greener approaches to extract bioactive compounds from natural resources. Pressurized hot water extraction (PHWE) is among the solvent free and efficient methods for extracting bioactive compounds. Experimental In this study, the suitability of PHWE for extracting bioactive compounds such as phenolics, hydrolysable tannins and flavonoids from Phyllanthus tenellus was investigated by UPLC-qTOF-MS. Results Solvent properties of water are significantly increased through imposing temperature at 121 °C and pressure at 15 p.s.i. Pressurized hot water extraction obtained 991-folds higher hydrolysable tannins than methanol extraction. Conclusion The extraction yields of hydrolysable tannins with PHWE was almost double of absolute methanol extraction.

Thiamine is known to be an important compound in human diet and it is a cofactor required for vital metabolic processes such as acetyl-CoA biosynthesis, amino acid biosynthesis, Krebs and Calvin cycle. Besides that, thiamine has been shown to be involved in plant protection against stress. In this study, the level of expression of THIC and THI1/THI4, the genes for the first two enzymes in the thiamine biosynthesis pathway were observed when oil palm (Elaeis guineensis) was subjected to oxidative stress. Primers were designed based on the consensus sequence of thiamine biosynthesis genes obtained from Arabidopsis thaliana, Zea mays, Oryza sativa, and Alnus glutinosa. Oxidative stress were induced with various concentrations of paraquat and samplings were done at various time points post-stress induction. The expression of THIC and THI1/THI4 genes were observed via RT-PCR and qPCR analysis. The expression of THIC was increased 2-fold, while THI1/THI4 gene transcript was increased 4-fold upon induction of oxidative stress. These findings showed that oil palm responded to oxidative stress by over-expressing the genes involved in thiamine biosynthesis. These findings support the suggestion that thiamine may play an important role in plant protection against stress.

The oil palm (Elaeis guineensis) is an important crop in Malaysia but its productivity is hampered by various biotic and abiotic stresses. Recent studies suggest the importance of signalling molecules in plants in coping against stresses, which includes thiamine (vitamin B1). Thiamine is an essential microelement that is synthesized de novo by plants and microorganisms. The active form of thiamine, thiamine pyrophosphate (TPP), plays a prominent role in metabolic activities particularly as an enzymatic cofactor. Recently, thiamine biosynthesis pathways in oil palm have been characterised but the search of novel regulatory element known as riboswitch is yet to be done. Previous studies showed that thiamine biosynthesis pathway is regulated by an RNA element known as riboswitch. Riboswitch binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA. TPP binds specifically to TPP riboswitch to regulate thiamine biosynthesis through a variety of mechanisms found in archaea, bacteria and eukaryotes. This study was carried out to hunt for TPP riboswitch in oil palm thiamine biosynthesis gene. Riboswitch detection software like RiboSW, RibEx, Riboswitch Scanner and Denison Riboswitch Detector were utilised in order to locate putative TPP riboswitch in oil palm ThiC gene sequence that encodes for the first enzyme in the pyrimidine branch of the pathway. The analysis revealed a 192 bp putative TPP riboswitch located at the 3’ untranslated region (UTR) of the mRNA. Further comparative gene analysis showed that the 92-nucleotide aptamer region, where the metabolite binds was conserved inter-species. The secondary structure analysis was also carried out using Mfold Web server and it showed a stem-loop structure manifested with stems (P1-P5) with minimum free energy of -12.26 kcal/mol. Besides that, the interaction of riboswitch and its ligand was determined using isothermal titration calorimetry (ITC) and it yielded an exothermic reaction with 1:1 stoichiometry interaction with binding affinities of 0.178 nM, at 30°C. To further evaluate the ability of riboswitch to control the pathway, exogenous thiamine was applied to four months old of oil palm seedlings and sampling of spear leaves tissue was carried out at days 0, 1, 2 and 3 post-treatment for expression analysis of ThiC gene fragment via quantitative polymerase chain reaction (qPCR). Results showed an approximately 5-fold decrease in ThiC gene expression upon application of exogenous thiamine. Quantification of thiamine and its derivatives was carried out via HPLC and the results showed that it was correlated to the down regulation of ThiC gene expression. The application of exogenous thiamine to oil palm affected ThiC gene expression, which supported the prediction of the presence of TPP riboswitch in the gene. Overall, this study provides the first evidence on the presence, binding and the functionality of TPP riboswitch in oil palm. This study is hoped to pave a way for better understanding on the regulation of thiamine biosynthesis pathway in oil palm, which can later be exploited for various purposes especially in manipulation of thiamine biosynthesis pathways in combating stresses in oil palm.

Thiamine (vitamin B1) is an essential microelement that is synthesised de novo by plants and microorganisms. The active form of thiamine is thiamine pyrophosphate (TPP), which plays a prominent role in plant's metabolic activity particularly as an enzymatic cofactor. In vivo analysis of thiamine in oil palm was performed where four months old oil palm seedlings were treated with 125 ml of 50 mM thiamine hydrochloride and tissue samples were collected at Day 0, 1, 2 and 3. The expression of ThiC gene fragment was analysed via quantitative real time polymerase chain reaction (qPCR) which showed an approximately five-fold decrease post-thiamine treatment. Analysis of thiamine and its derivatives via high performance liquid chromatography (HPLC) showed that the concentration of thiamine decreased, while TPP increased post-thiamine treatment. It is suggested that thiamine biosynthesis in oil palm can be effectively regulated at the physiological concentrations of the vitamin.

Aims: The use of microalgae as source of natural antioxidants is under explored in Malaysia. Previous studies have shown that microalgae contain minerals, polysaccharides, amino derivatives, carotenoids and phenolic compounds. This study aimed to determine total phenolic and flavonoid compounds and antioxidant activity when microalgae (Nannochloropsis oculata and Tetraselmis sp.) and cyanobacterium (Anabaena sp.) were subjected to abiotic stresses. Methodology and results: Treatment of sodium chloride (NaCI), sodium hypochlorite (NaOCI) and copper (Cu2+) were given when the cultures reached the exponential phase of growth and were collected at three different time points. Nontreated cultures were used as controls. Total phenolic and flavonoid contents were determined using Folin-Ciocalteau phenol reagent and aluminium chloride colorimetric assays. Antioxidant activities were measured using 2,2-diphenyl-1- picrylhydrazyl (DPPH) free radical scavenging activity assay. Tetraselmis sp. exhibited the highest phenolic content under copper stress (10.35 ± 0.33 µg GAE/mg extract). Nannochloropsis oculata showed the highest total flavonoid content under copper stress (33.85±3.16 µg QE/mg extract). Anabaena sp. showed the highest radical scavenging activity under NaOCI stress (96.42 ± 0.26%). Conclusion, significance and impact of study: This study showed that total phenolic, flavonoid and antioxidant activities in treated cultures were high compared to non-treated cultures. These microorganisms could be utilized as a source of useful bioactive compounds while exploiting its abundance.